An investigation on conjugated linoleic acid content, fatty acid composition, and physicochemical characteristics of Iranian Kurdish butter oil

Abstract In this study, physicochemical and quality properties, fatty acid composition, and triglyceride composition of Iranian Kurdish butter oil (IKBO) obtained from yogurt drink (doogh) butter were investigated. Local doogh butter, prepared from cow's (CIKBO) and ewe's milk (EIKBO), was utilized as the raw material for this purpose. The free fatty acids (FFA) and peroxide values of IKBOs of the cow (CIKBO) and ewe (EIKBO) were obtained at 0.41 ± 0.01 and 0.39 ± 0.01 (g Oleic acid 100/g oil), and 1.32 ± 0.00 and 1.35 ± 0.00 (meq O2 kg/oil), respectively. The amounts of saturated fatty acids (SFAs): 70.27 ± 0.62 and 72.13 ± 0.84 (g/100 g), monounsaturated fatty acids (MUFAs): 19.37 ± 0.74 and 20.56 ± 0.97 (g/100 g), and polyunsaturated fatty acids (PUFAs): 1.22 ± 0.12 and 2.75 ± 0.38 (g/100 g) were obtained in CIKBO and EIKBO, respectively. The significant majority of the fatty acids (FAs) in the examined CIKBO and EIKBO were myristic (CIKBO: 13.76 ± 0.02 (g/100 g) and EIKBO: 14.83 ± 0.07 (g/100 g)), palmitic (CIKBO: 33.14 ± 0.28 (g 100/g) and EIKBO: 31.86 ± 0.02 (g/100 g)), stearic (CIKBO: 8.27 ± 0.06 (g/100 g) and EIKBO: 7.95 ± 0.06 (g/100 g)), capric (CIKBO: 4.83 ± 0.03 (g/100 g) and EIKBO: 6.75 ± 0.01 (g/100 g)), and oleic acids (CIKBO: 15.37 ± 0.12 (g/100 g) and EIKBO: 17.83 ± 0.02 (g/100 g)). The average of conjugated linoleic acid (CLA) content in EIKBO (2.20 ± 0.22 (g/100 g)) was higher than that in CIKBO (0.92 ± 0.25 (g/100 g)) (p < .05). Therefore, EKIBO is considered the superior natural supply of CLA.

; however, there is no publication on the properties of IKBO (Kurdish Roghan).
Due to their high saturated fatty acids (SFAs) content, dietary milk fats have always been related to arteriosclerosis and coronary heart disease (Grundy, 1997;Lokuruka, 2007). Nonetheless, current research has concentrated on the beneficial components of milk lipids, including conjugated linoleic acid (CLA) (Crumb & Vattem, 2011;Nagpal et al., 2012). The nutritional benefits of CLA consumption have been demonstrated in vitro and animal studies. These benefits include anticarcinogenic characteristics (Parodi, 1997), antiadipogenic characteristics (House et al., 2005), reduction of LDL cholesterol and heart disease (Crumb & Vattem, 2011), and lean body mass enhancer (Park et al., 1997). CLA refers to a blend of linoleic acid's conjugated positional and geometric isomers (cis-9, cis-12 octadecadienoic acid).
Butter and ghee are the largest sources of cis-9 and trans-11octadecadienoic acid, and it has been claimed that their intake may alter CLA levels in blood serum and human milk (Parodi, 1994;Sindhuja et al., 2020). Through the biohydrogenation pathway's isomerase and reductase enzymes, microbial fermentation leads to an increase in CLA content during ghee production (Aneja & Murthi, 1990;Mehta, 2009).
The milk's fatty acid content is critical for processing and human health (Kay et al., 2005). Some studies are being conducted in several countries on the CLA content and the composition of FAs in dairy products (Donmez et al., 2005;Guler et al., 2010;Indu & Jayaprakasha, 2021;Kelly et al., 1998;Ledoux et al., 2005). The CLA content and the composition of FAs of IKBO are unknown.
Therefore, this study aimed to evaluate the physicochemical and quality properties, fatty acid composition, and triglyceride composition of Iranian Kurdish butter oil (IKBO) (the traditional and beneficial oil) obtained from yogurt drink butter named doogh, which is prepared from cow's and ewe's milk (as the raw material).

| Raw milk
Fresh raw milk (cow and ewe) was sourced in Kurdistan, Iran's northwest region. The physicochemical quality properties of raw milk were determined according to the described methods by the International Organization for Standardization (ISO), including titratable acidity (ISO 660, 2020), dry matter (Uzatici & Yayintaş, 2017), fat content (ISO 19662, 2018), and pH (ISO 26323, 2009). Yogurt was applied as the starting culture produced in the laboratory. All studies were conducted at the dairy pilot plant in the department of food science and technology of the University of Tehran, Iran.

| Butter oil production
To get butter oil samples, the following technique was performed.
Raw whole milk (cow and ewe) was utilized to make yogurt without additional processing. The fresh milk was heated to 90°C for 15 min, inoculated with the starting culture, and incubated at 40-42°C until the pH reached 4.6-4.7, at a certain point, then cooled to 4-5°C to end the fermentation. Next, the yogurt samples were kept at 4-5°C for 12-14 h to crystallize the milk fat. Subsequently, enough hot water (1:1 vol) was added to obtain the desired doogh (Iranian yogurt drink) and churned at 14-16°C for about 30 min by a pilot churning machine (Model 2585 Crealan Package (CP), Chicago). The buttermilk was removed after the churning process, and the butter grains were rinsed twice with cold water (7-9°C) to eliminate any remaining buttermilk. Fresh butter samples (ewe and cow) were put in glazed pots and rubbed with an appropriate quantity of dry salt (10% w/w) before being kept at 18 ± 2°C for 4 days. The samples were returned multiple times over this period.
After this first interval, dry salt was applied to cover the butter samples thoroughly, forming a continuous salt coating throughout the whole butter surface. The pots were then carefully covered and kept at 18 ± 2°C for 4 months to mature. The samples were then melted at 90 °C and separated into oil and serum phases. At 110°C, the melted butter samples were swirled until the oil became a light brown hue. The oil phase was clarified by filtering it through multiple layers of cheesecloth and storing it at −24°C until further analysis. Two distinct sets were prepared for analysis using the technique outlined above.

| Physicochemical properties of butter oil
The fat content of obtained butter oil was measured using Internationaler (1966); Codex Standard (1999); amended 2018.
Briefly, butter oil samples were warmed and melted in a water bath at 40-50°C till the water separated. After 5 min of centrifugation at 3500 rpm, the transparent supernatant was filtered using filter paper.

Determination of the peroxide value
The peroxide values of the obtained fat from butter oil samples were determined according to the described method in ISO 3976 (2006).

Determination of the free fatty acids (FFAs)
The FFAs' values of the obtained fat of butter oil samples were measured according to ISO 660 (2020).

| Determination of fatty acid composition
The butter oil samples were immediately warmed at 45°C and  (Fontecha et al., 1998).

| Statistical analysis
Data analysis was gained at least in triplicates and averaged. Oneway analysis of variance (ANOVA) was used for statistical analysis.  Table 1 summarizes the qualities of fresh raw milk samples used as the raw material for preparing IKBO. Physicochemical characteristics, including pH, TA, dry matter (DM), and fat contents of raw milk used for the preparation of butter oil, are presented in Table 1.

| RE SULTS AND D ISCUSS I ON
These qualities are determined by physiochemical parameters and compliance with sanitary-hygienic milking requirements. In order to determine the needed features, numerous tests are available (for instance, titratable acidity (TA) is a quick test (90 s to complete) that indicates the quality of raw milk and offers an indirect assessment of the acid level in milk).
Total saturated fatty acids (g 100/g oil) were found to be 70.27 ± 0.62 and 72.13 ± 0.84 (g 100/g oil) in both CIKBO and EIKBO samples, respectively, which was more than overall unsaturated fatty acids, ranged from CIKBO: 20.59 ± 0.27 to EIKBO: 23.31 ± 0.24 (g 100/g oil). Overall unsaturated fatty acids are divided into total monounsaturated fatty acids (MUFA) and total polyunsaturated fatty acids (PUFA). These findings corroborate prior research on various butter oils derived from cow's milk.
In a study, the saturated fatty acids (SFAs) content of "Fulani butter oil" derived from cow's milk was reported at 53.3 (g 100/g oil), in which the palmitic acid is the predominant fatty acid (30.2 (g 100/g oil)) (Glew et al., 1999). Also, in the other study, the SFAs in butter oil generated from cow and ewe milk obtained 67.06 (g 100/g oil) and 69.10 (g 100/g oil), respectively, and palmitic acid was the predominant fatty acid in both butter oils (Sağdıç et al., 2004).
Additionally, the SFAs value of butter oil derived from buffalo milk was obtained at 70.72 (g 100/g oil), and palmitic acid was the major fatty acid (31.89 (g 100/g oil)) (Fatouh et al., 2007). The SFAs content of butter oil generated from ewe's milk was obtained at 59.13 (g 100/g oil), with oleic acid being the predominant fatty acid (31.08 (g 100/g oil)) (Özkanlı & Kaya, 2007) which oleic acid value was further than that obtained in this study. The concentration of SFAs in ewe's milk butter oil (72.13 (g 100/g oil)) in this study was much more significant than those in previous reports by  (Sağdıç et al., 2004).
Also, the MUFA level of butter oil produced from cow's milk (19.37 (g 100/g butter oil)) was less than previous reports by Dhibi et al. (2013) and Samet-Bali et al. (2009).
Prior studies showed that the MUFA amount of the total fatty acids in Fulani butter oil was 32 (g 100/g butter oil), which is greater than the value observed in this study (Glew et al., 1999).
The PUFAs level of Fulani butter oil was 3.32 (g 100/g oil), which is greater than the current study's results (Glew et al., 1999).
The total TUFAs levels of the CIKBO and EIKBO samples were significantly greater than those reported by Sağdıç et al. (2004) and Dhibi et al. (2013). In this study, the mean total CLA values of the CIKBO and EIKBO datasets were 0.98 ± 0.04 (g 100/g butter oil) and 2.20 ± 0.10 (g 100/g butter oil), respectively, of total fatty acids. CLA c9, t11 was the predominant CLA isomer in all cases.
Prior investigations revealed that the overall CLA content of sheep milk fat is much greater than that of cow or goat milk fat at 1.08 (g 100/g fat), 1.01(g 100/g fat), and 0.65 (g 100/g fat), respectively. The animal diet, the specific attributes of the milk used to produce butter oil, with particular reference to the species and CLA content of the milk, as well as the processing and manufacturing techniques, have a substantial impact on the quantity of CLA in dairy products, which vary seasonally owing to variabilities in feeding aspects (Chilliard et al., 2003;Prandini et al., 2007;Sindhuja et al., 2020). The most extensive seasonal variations of CLA were seen in sheep milk, which ranged from 1.28 (g 100/g oil) in the summer to 0.54 (g 100/g oil) at the end of the winter (Indu & Jayaprakasha, 2021).
The CLA (C18:2 c9t11) amounts of various Turkish kinds of cheese prepared using conventional techniques ranged between 0.44 and 1.04 (g 100/g oil) (Donmez et al., 2005). The CLA content and the composition of fatty acids of several Turkish dairy products (butter, processed cheese, Kaymak, and cream) were studied, and it discovered that the CLA amounts ranged from 0.97 to 1.05 (g 100/g fat) and that rumenic acid was detected as the predominant CLA isomer in Turkish dairy products (Seçkin et al., 2005). The total CLA

ACK N OWLED G M ENT
The authors express their thanks to the research deputy of the University of Tehran and Standard Research Institute, Karaj, for their facilitation support.

FU N D I N G I N FO R M ATI O N
This work is not supported with research funding.

CO N FLI C T O F I NTE R E S T
Authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.